These practical protocols are designed for students doing an Extended Project Qualification, Advanced Higher investigation, or IB investigation in biology.
They are linked to our Medical matters – fungal infections – project starter.
Possible areas for investigation
- Investigate different types of yeast with particular antifungals
- Investigate different types of antifungals with one yeast species – could use medical products, veterinary products, herbal product and natural products
- Compare treatments for thrush with treatments for athletes foot – some products allow growth of the yeast in the inhibition zone after a few days
- 1 gram of yeast
- Bottle of 10 cm3 of sterile distilled water
- 2 sterile syringes or pipettes
- sterile spreader
- number 4 or 5 cork borer
- sample of anti-fungal treatment
- 2 glucose yeast agar plates
- sterile seeker
- sterile forceps
- innoculating loops
- Add one gram of yeast to a bottle of 10 cm3 of sterile distilled water and shake thoroughly.
- Alternatively add a few loopfuls of yeast from a pre-prepared culture to 2 cm3 of sterile distilled water and shake thoroughly.
- Warm this solution to approx 40°
- Pre-label your agar plate with your initial and the date and stating species of yeast and treatment to be used.
- Use a sterile syringe to add 0.3 cm3 of the yeast solution to the agar plate and spread with a sterile spreader. (the spreader can be sterilised by placing in ethanol, flaming and allowing to burn off)
- Flame a 4 or 5 cork borer, use it to make a well in the agar plate and remove thecircle of agar with a sterile seeker.
- Add 0.1 cm3 of anti-fungal treatment to the well
- Seal plate and incubate for 3 days
- Measure the diameter of any area of inhibition over the next 3 to 4 days
General equipment list
For a project to investigate fungal inhibition, you are likely to need the following equipment and materials
- Yeast samples dried, freeze dried or fresh
- Sterile distilled water
- Sterile syringes or pipettes
- Number 4 or 5 cork borer
- sample of anti-fungal treatment
- Glucose yeast agar plates
- Petri dishes
- Sterile seeker
- Sterile forceps
- Inoculating loops
Preparation of Materials
Seekers and forceps can be sterilised by wrapping them in tin-foil and then autoclaving or the students can be taught to alcohol flame them.
Several different yeast preparations can be used:
Saccharomyces cerevisiae can be used taken from a block of yeast.
Phaffia rhodozyma can be grown up on a slope or agar plate.
Both yeasts will grow on Glucose yeast agar ( Phaffia grows at 20°C and Saccharomyces at 35°C.)
Although it would not be possible to use Candida albicans for this investigation Candida utilis is available from National Collection of Yeast Cultures.
Antifungal products can be bought from a chemist shop – Canistan; Daktarin; Diflucan etc. Antifungal Vet products can be bought in pet shops.
Making Glucose Yeast Agar – (this method will make enough agar for 3 petri dishes)
- 100 cm3 beaker
- stirring rod
- 3 McCartney bottles
- weighing boat
- yeast extract
- 0.1M Sulphuric Acid
- distilled water
- balance (accurate to 0.5g)
- pH paper
- 10ml syringe
- Weigh out 1g of glucose and 0.5g of yeast extract.
- Place the chemicals together in a beaker and add enough distilled water to make up to the 50 cm3 mark on the beaker.
- Stir thoroughly and then test the pH. Add enough sulphuric acid one drop at a time to make the solution pH6 – test with pH probe or pH paper.
- Place 15 cm3 of this solution into a McCartney bottle- you should have enough for 3 bottles.
- Add 0.3g of agar to each McCartney bottle.
- Shake and then place lid loosely on bottle and initial your bottles.
- Autoclave for 15 minutes.